The Black Sea is the largest semi‐closed permanently anoxic basin on our planet with long‐term stratification. The study aimed at describing the Black Sea microbial community taxonomic and functional composition within the range of depths spanning across oxic/anoxic interface, and to uncover the factors behind both their vertical and regional differentiation. 16S rRNA gene MiSeq sequencing was applied to get the data on microbial community taxonomy, and the PICRUSt pipeline was used to infer their functional profile. The normoxic zone was mainly inhabited by primary producers and heterotrophic prokaryotes (e.g., Flavobacteriaceae, Rhodobacteraceae, Synechococcaceae) whereas the euxinic zone—by heterotrophic and chemoautotrophic taxa (e.g., MSBL2, Piscirickettsiaceae, and Desulfarculaceae). Assimilatory sulfate reduction and oxygenic photosynthesis were prevailing within the normoxic zone, while the role of nitrification, dissimilatory sulfate reduction, and anoxygenic photosynthesis increased in the oxygen‐depleted water column part. Regional differentiation of microbial communities between the Ukrainian shelf and offshore zone was detected as well, yet it was significantly less pronounced than the vertical one. It is suggested that regional differentiation within a well‐oxygenated zone is driven by the difference in phytoplankton communities providing various substrates for the prokaryotes, whereas redox stratification is the main driving force behind microbial community vertical structure.